External morphogenesis of the tardigrade Hypsibius dujardini as revealed by scanning electron microscopy

Tardigrada, commonly called water bears, is a taxon of microscopic panarthropods with five‐segmented bodies and four pairs of walking legs. Although tardigrades have been known to science for several centuries, questions remain regarding many aspects of their biology, such as embryogenesis. Herein, we used scanning electron microscopy to document the external changes that occur during embryonic development in the tardigrade Hypsibius dujardini (Eutardigrada, Parachela, Hypsibiidae). Our results show an accelerated development of external features, with approximately 30 hrs separating the point at which external structures first become recognizable and a fully formed embryo. All segments appear to arise simultaneously between ∼20 and 25 hrs of development, and no differences in the degree of development could be detected between the limb buds at any stage. Claws emerge shortly after the limb buds and are morphologically similar to those of adults. The origin of the claws is concurrent with that of the sclerotized parts of the mouth, suggesting that all cuticular structures arise simultaneously at ∼30 hrs. The mouth arises as an invagination in the terminal region of the head at ∼25 hrs, closes later in development, and opens again shortly before hatching. The anlagen of the peribuccal lobes arise as one dorsal and one ventral row, each consisting of three lobes, and later form a ring in the late embryo, whereas there is no indication of a labrum anlage at any point during development. Furthermore, we describe limited postembryonic development in the form of cuticular pores that are absent in juveniles but present in adults. This study represents the first scanning electron micrographs of tardigrade embryos, demonstrating the utility of this technique for studying embryogenesis in tardigrades. This work further adds an external morphological perspective to the developmental data already available for H. dujardini , facilitating future comparisons to related panarthropod taxa. J. Morphol. 278:563–573, 2017. © 2017 Wiley Periodicals, Inc.     

Further study of the embryonic development of Oreochromis niloticus (Cichlidae, Teleostei) using scanning electron microscopy

The chronology of development of Oreochromis niloticus embryos was examined by scanning electron microscopy (S.E.M.) and the external structures which evolved during the embryonic period were analysed. Twenty-four stages of embryonic development of this fish at 25°C are described with emphasis on the cleavage phases and morphogenesis of external organs and tissues. Because the development of internal organs could not be observed under S.E.M., the necessary investigation was completed by light microscopy.     

Morphometric study of embryonic development of Macrobrachium borellii (Arthropoda: Crustacea)

Summary

Embryo morphometry and developing time from just-laid eggs until hatching were described in the palaemonid prawn, Macrobrachium borellii, using a rapid non-invasive staging method. Embryos were kept in the laboratory under controlled conditions and development divided into seven stages according to major morphological characteristics. This lecithotrophic, freshwater shrimp has a highly abbreviated type of hatching development after 39 ± 2 days as postlarvae at 24°C. Morphometry was recorded using a stereoscopic microscope with an image analyzer. Area, perimeter, maximum and minimum diameters and shape were measured in yolk sac, egg-coat and eye, respectively, and they were statistically selected as the best to define the stages. Eggs are ovoid with a maximum diameter that varies from the moment of oviposition to the time of hatching from 1.5 to 2.0 mm, respectively. Water content and egg size increase along with development, whereas egg shape only varies just before hatching when the egg becomes strongly ovoid. Egg coat and eye variables significantly increase as the embryo develops while all yolk variables decrease as the embryo consumes the vitellus. Yolk represents more than 95% of the egg at the time of oviposition, falling to 22% by the time of hatching. The major yolk area decrease is observed between stages 4 and 5, which is coincident with a marked increase in the catabolism. Using only egg coat, yolk and eye shape and maximum diameter, a researcher can straightforwardly identify a developing stage with an accuracy ranging from 70 to 100%. This tool may be employed in other species provided they have transparent chorion.     

The (not very) typical protonymphons of Pycnogonum litorale

Sea spiders are unique and poorly known marine chelicerates. Their larvae are even less studied, especially at the ultrastructural level. Here, we examined the hatchlings of Pycnogonum litorale (Strøm, 1,762) using histology, SEM and TEM. Existing classifications place these larvae among “typical” protonymphons, together with Nymphon brevirostre. Our results, however, revealed major differences between the two species. Hatchlings of P. litorale are endotrophic for 1–2 weeks, with yolk deposits in the body wall and a reduced secretory apparatus. They lack a body cavity, demonstrate an unusual modification of the midgut sheath cells and a complex subesophageal ganglion, which includes neuromeres of the prospective walking legs 1. These larvae also possess well-developed glia and complex sensory structures: eyes, V-shaped mechanoreceptive bristles, integrated chemo- and mechanoreceptors, and three types of concealed mechanoreceptors embedded into the body wall and only seen on the sections. In this paper we also propose a new interpretation of the pycnogonid larval types: we present a set of traits useful for diagnosis and a preliminary classification. Finally, we discuss the complexity of glial types in sea spiders and other arthropods.     

Gills of the medaka (Oryzias latipes): A scanning electron microscopy study

The general morphology and surface ultrastructure of the gills of adult and larvae medaka (Oryzias latipes) were studied in freshwater and seawater using scanning electron microscopy. The gills of all examined fish were structurally similar to those of other teleosts and consisted of four pairs of arches supporting (i) filaments bearing lamellae and (ii) rakers containing taste buds. Three cell types, specifically pavement cells, mitochondria‐rich cells (MRCs), and mucous cells, constituted the surface layer of the gill epithelium. Several distinctive characteristics of medaka gills were noted, including the presence of regularly distributed outgrowth on the lamellae, enlarged filament tips, the absence of microridges in most pavement cells in the filament and lamellae and the presence of MRCs in the arch at the filament base. A rapid mode of development was recorded in the gills of larval fish. At hatching, the larvae already had four arches with rudimentary filaments, rakers, and taste buds. The rudimentary lamellae appeared within 2 days after hatching. These results suggest the early involvement of larval gills in respiratory and osmoregulation activities. The responses of the macrostructures and microstructures of gills to seawater acclimation were similar in larvae and adult fish and included modification of the apical surface of MRCs, confirming the importance of these cells in osmoregulation. The potential roles of these peculiarities of the macrostructures and microstructures of medaka gills in the major functions of this organ, such as respiration and osmoregulation, are discussed.     

Stages of embryonic development in the amphipod crustacean, Parhyale hawaiensis

Studying the relationship between development and evolution and its role in the generation of biological diversity has been reinvigorated by new techniques in genetics and molecular biology. However, exploiting these techniques to examine the evolution of development requires that a great deal of detail be known regarding the embryonic development of multiple species studied in a phylogenetic context. Crustaceans are an enormously successful group of arthropods and extant species demonstrate a wide diversity of morphologies and life histories. One of the most speciose orders within the Crustacea is the Amphipoda. The embryonic development of a new crustacean model system, the amphipod Parhyale hawaiensis, is described in a series of discrete stages easily identified by examination of living animals and the use of commonly available molecular markers on fixed specimens. Complete embryogenesis occurs in 250 h at 26 degrees C and has been divided into 30 stages. This staging data will facilitate comparative analyses of embryonic development among crustaceans in particular, as well as between different arthropod groups. In addition, several aspects of Parhyale embryonic development make this species particularly suitable for a broad range of experimental manipulations.     

Morphological phylogenetics of the sea spiders (Arthropoda: Pycnogonida)

Pycnogonids or sea spiders are a group of marine arthropods whose relations to the chelicerates have been an issue of controversy. Higher-level phylogenetic relationships among the lineages of sea spiders are investigated using 36 morphological characters from 37 species from all extant families and a Devonian pycnogonid fossil. This is one of the first attempts to analyze the higher-level relationships of the Pycnogonida using cladistic techniques. Character homoplasy (implied weights) is taken into account to construct a polytomous, most-parsimonious tree in which two major clades within Pycnogonida are obtained. Clade A includes Ammotheidae paraphyletic with Colossendeidae, Austrodecidae and Rhynchothoracidae, and clade B is formed by Nymphonidae, Callipallenidae (apparently paraphyletic), Pycnogonidae and Phoxichilidiidae. The analysis of equally weighted data is presented and helps to identify those characters less consistent. The reduction of the chelifores, palps and ovigers — shown independently within each of the clades as parallel evolution events — challenges the assumption of a gradual mode of reduction within the group, according to analysis of unordered vs ordered characters. Most of the phylogenetic affinities proposed here are compatible with traditional classifications. However, traditional taxonomic characters need to be complemented by sets of anatomical, molecular and developmental data, among others, to produce more robust phylogenetic hypotheses on the higher- and lower-level relationships of the sea spiders.     

A new species of Hedgpethia (Arthropoda, Pycnogonida, Colossendeidae) from southwestern Japan

We describe Hedgpethia spinosasp. n. based on a single male specimen obtained from 197-207 m depth, south of Yaku Island, Kagoshima Prefecture, Japan. Among 15 previously known congeners, the new species resembles Hedgpethia bicornis (Losina-Losinsky & Turpaeva, 1958), Hedgpethia chitinosa (Hilton, 1943), and probably Hedgpethia brevitarsis (Losina-Losinsky & Turpaeva, 1958), in having a mid-dorsal tubercle on the posterior rim on each trunk segment. The new species, however, is distinguishable from those by a pair of horns on the anterior margin of the cephalic segment, spines on the first coxae, and denticulate spines on the strigilis. The new species represents the fifth member of the genus so far known from Japanese waters, in addition to Hedgpethia brevitarsis (Losina-Losinsky & Turpaeva, 1958), Hedgpethia chitinosa (Hilton, 1943), Hedgpethia dofleini (Loman, 1911), and Hedgpethia elongata Takahashi, Dick & Mawatari, 2007.     

Observation of the embryonic development in Pseudoplatystoma coruscans (Siluriformes: Pimelodidae) under light and scanning electron microscopy

Pseudoplatystoma coruscans is a very popular species for tropical fish culture as it has boneless meat of delicate taste and firm texture. Few studies on fish reproductive biology refer to the morphological features of eggs. The goal, therefore, of this present work was to perform a structural and ultrastructural analysis of fertilization and embryonic development in P. coruscans. The incubation period, from fertilization to hatching, lasts 13 h at 28/29 degrees C and 18 h at 27 degrees C. The oocytes had a mean diameter of 0.95 mm and hatched larvae were 2.55 mm in diameter. Analysing their development, we observed round, yellow oocytes that bore a double chorion membrane and a single micropyle. At 10 s after fertilization, several spermatozoa were detected attached to the oocyte surface. After 1 min of development, a fertilization cone that obstructed the micropyle could be observed. Segmentation started between 20 and 30 min after fertilization, when the egg cell was then formed. The first cleavage occurred between 30 and 45 min after fertilization, prior to reaching the morula stage (75 and 90 min after fertilization). The epiboly movement started at 120 and 180 min after fertilization and ended at 360 and 480 min after fertilization. Differentiation between cephalic and caudal region was detected after 420 and 600 min after fertilization and larvae hatched between 780 and 1080 min after fertilization. Seven main embryonic development stages were identified: egg cell, cleavage, morula, blastula, gastrula, segmentation with differentiation between cephalic and caudal regions, and hatching.     

Polarimetric optical scanning microscopy of zebrafish embryonic development using the coherency matrix

Many of the most important resolution improvements in optical microscopy techniques are based on the reduction of scattering effects. The main benefit of polarimetry-based imaging to this end is the discrimination between scattering phenomena originating from complex systems and the experimental noise. The determination of the coherency matrix elements from the experimental Mueller matrix can take advantage of scattering measurements to obtain additional information on the structural organization of a sample. We analyze the contrast mechanisms extracted from (a) the coherency matrix elements, (b) its eigenvalues and (c) the indices of polarimetric purity at different stages of zebrafish embryos, based on previous work using Mueller matrix optical scanning microscopy. We show that the use of the coherency matrix and related decompositions leads to an improvement in the imaging contrast, without requiring any complicated algebraic operations or any a priori knowledge of the sample, in contrast to standard polarimetric methods.     

A study by scanning (SEM) and transmission (TEM) electron microscopy of the glomerular capillaries in developing rat kidney

Summary Kidneys of 2 to 10 day-old rats of Wistar and Sprague-Dawley strains were fixed with glutaraldehyde by retrograde vascular perfusion and then prepared for observation in TEM and SEM. In addition methacrylate casts of differentiating glomerular capillaries were examined by SEM. Although the glomerular vascular pattern differs from one glomerulus to another, its differentiation proceeds according to the following general plan. First the glomerular capillary splits longitudinally, finally to form 3 to 5 lobules consisting of a capillary network, sustained centrally by the mesangium.In the present study the differentiation of glomerular capillaries is described in five successive arbitrarily selected stages. At Stage I a capillary loop penetrates between the lower limb and the middle segment of the S-shaped body, the rudimentary nephron. At Stage II the capillary undergoes a first subdivision, establishing the primitive lobulation of the glomerulus. At Stage III the vascular and urinary poles differentiate. At Stage IV the glomerulus assumes the aspect of a spherical body, and the capillaries in each lobule undergo subdivision. In Stage V the glomerular vascular pattern approaches its adult appearance, although the maturation processes continue for an extended period of time. Hence in the 10 day-old rat the best-differentiated glomeruli are half the size of adult glomeruli, and their capillary loops are proportionally less well-developed. The capillaries of adjacent lobules may communicate with each other, but a direct vascular shunt between the afferent and efferent vessels cannot be demonstrated.Presented in part at the 60ème Congrés de l'Association des Anatomistes in Nice (27–30 May 1976) and the 73. Versammlung der Anatomischen Gesellschaft in Innsbruck (12–15 September 1978)The author thanks Miss Morena Ghisletta for excellent technical assistance     

Morphogenesis of Pseudopallene sp. (Pycnogonida, Callipallenidae) I: embryonic development

Embryonic development of Pycnogonida (sea spiders) is poorly understood in comparison to other euarthropod lineages with well-established model organisms. However, given that pycnogonids potentially represent the sister group to chelicerates or even to all other euarthropods, their development might yield important data for the reconstruction of arthropod evolution. Using scanning electron microscopy, fluorescent nucleic staining and immunohistochemistry, the general course of embryonic morphogenesis in Pseudopallene sp. (Callipallenidae), a pycnogonid with prolonged embryonic development, is described. A staging system comprising ten stages is presented, which can be used in future studies addressing specific developmental processes. The initially slit-like stomodeum anlage forms at the anterior end of an eight-shaped germ band and predates proboscis outgrowth. The latter process is characterized by the protrusion of three cell populations that are subsequently involved in pharynx formation. In later stages, the proboscis assumes distally a horseshoe-like shape. At no time, a structure corresponding to the euarthropod labrum is detectable. Based on the complete lack of palpal and ovigeral embryonic limbs and the early differentiation of walking leg segments 1 and 2, the existence of an embryonized protonymphon stage during callipallenid development is rejected. The evolution of pycnogonid hatching stages, especially within Callipallenidae and Nymphonidae, is re-evaluated in the light of recent phylogenetic analyses. Specifically, the re-emergence of the ancestral protonymphon larva (including re-development of palpal and ovigeral larval limbs) and a possible re-appearance of adult palps in the nymphonid lineage are discussed. This challenges the perception of pycnogonid head appendage evolution as being driven by reduction events alone.     

The deep-water pycnogonids (Arthropoda: Pycnogonida) of the northeastern Atlantic Ocean

Over 2000 specimens of pycnogonid from deep-sea collections made in the northeastern Atlantic Ocean by IOS and SMBA between 1971 and 1994 have been analysed. Thirty-five species were identified, two being new to science, viz Nymphon akanthochoeros and an unnamed Anoplodactylus species. The deep-sea Pycnogonida of the northeastern Atlantic are reviewed using these data and records from the literature. Keys are presented for the six families represented by more than one species. The pycnogonid fauna of this area is characterized particularly by Paranymphon spinosum and Colossendeis clavata , with other dominant species including Colossendeis species, notably C. macenima , and Nymphon laterospinum, Anoplodactylus typhlops and A. arnaudae. Of the 54 species recorded below 200 m in the northeastern Atlantic, available evidence suggests that nine are 'endemic'. Relative distributions of the other species are discussed. A distinct change in the pycnogonid fauna with depth was found at around 1400 m. There is latitudinal separation of species groupings, shown most conspicuously by the shallower water species.     

A new species of Hemichela Stock, 1954 from the South China Sea (Arthropoda,Pycnogonida, Ammotheidae)

A new species of pycnogonid collected by the Chinese research vessel, R/V HY IV, during deep sea cruises to the South China Sea in 2013, is described. The new species, Hemichela nanhaiensis, obtained from more than 1300 m depth, is distinguished from the other two species in the genus by the characters of the chela dactylus with 12 denticulations on the inner margin and by the presence of taller tubercles on the lateral processes.     

Postembryonic development of the female reproductive system in the pycnogonid Propallene longiceps (Pycnogonida,Callipallenidae)

The postembryonic development of the female reproductive system in the pycnogonid Propallene longiceps is examined. The germ cells can be detected first in the later stage of the 3rd instar and become a paired gonad covered with gonadal epithelium in the next instar. The larval gonad changes its shape: paired at the 4th instar, reversed U-shaped at the fifth, unpaired at the sixth, and paired again at the seventh. Oocytes can be distinguished, and the extension of the ovary into the walking legs begins at the 7th instar. Growing oocytes protrude outward from the ovary on cellular stalks in the pedal part. The trunk ovary becomes U-shaped, and the oviducts and genital pores start forming at the 8th instar. The disappearance of trunk ovary begins at the 9th instar, and is complete at the next adult stage. The connection between the pedal ovarian lumen and the genital pores via the oviducts is complete in the adult, and the female reproductive system becomes segmentally arranged. This study confirms that the segmental arrangement of adult female reproductive system in P. longiceps, which is unique among recently described arthropods, is a secondary state in pycnogonids attained by reducing the trunk part of ovary.     

A combined light and scanning electron microscopy study

The uppermost Eocene Florissant Formation, Rocky Mountains, Colorado, has yielded numerous insect, vertebrate and plant fossils. Three previous comprehensive palynological studies investigated sections of lacustrine deposits of the Florissant Formation and documented the response of plant communities to volcanic eruptive phases but overall found little change in plant composition throughout the investigated sections. These studies reported up to 150 pollen and spore phenotypes. In the present paper, we used a taxonomic approach to the investigation of dispersed pollen and spores of the Florissant Formation. Sediment samples from the shale units containing macrofossils were investigated using light microscopy (LM) and scanning electron microscopy (SEM). The general picture of the palynoflora is in agreement with previous studies. However, the combined LM and SEM investigation provides important complementary information to previous LM studies. While a fairly large amount of previous pollen determinations could be confirmed, the purported taxonomic affinities of several pollen phenotypes need to be revised. For example, pollen referred to as Podocarpus or Podocarpidites sp. belongs to the Pinaceae Cathaya, Malus/Pyrus actually belongs to Dryadoideae, pollen of the form genus Boehlensipollis referred to as Proteaceae/Sapindaceae/Elaeagnaceae or Cardiospermum belongs to Sapindaceae but not to Cardiospermum, and pollen of Persicarioipollis sp. B with previously assumed affinities to Polygonaceae actually belongs to Thymelaeaceae. Pandaniidites and one type of Malvacipollis cannot be linked with Pandanaceae and Malvaceae. A few taxa are new records for Florissant (Ebenaceae: Diospyros; Mernispermaceae; Trochodendraceae: Tetracentron). In general, SEM investigations complement the LM palynological studies and improve the identification of dispersed pollen and spores and enable integration of data from dispersed fossil pollen into a wide range of comparative morphological, taxonomic, evolutionary, biogeographic and phylogenetic studies.     

Larval development of the rhizocephalan cirripede Briarosaccus tenellus (Maxillopoda: Thecostraca) reared in the laboratory: a scanning electron microscopy study

SEM investigations of laboratory-reared larvae of Briarosaccus tenellus Boschma, 1970, revealed five naupliar instars, one more than previously described for the Rhizocephala. The external features of these and the cypris larva are described in detail. The youngest instar (NO) is characterised by a better developed furca than in subsequent stages and one additional antennulary seta. Differences in outline, shape of antennulae, and hind-body denticulation, offer the potential of individual discrimination. All the nauplii possess a transparent, hollow cuticular ring around their body. This flotation collar represents the bulged margin of the larval head shield and is attached to the body along a delicate ridge. Three pairs of tiny pores in contact with the ridge possibly regulate inflation of the ring, but details of this mechanisms remain unknown. Due to total lecithotrophy, the nauplii of B. tenellus have limbs setation reduced to that needed for swimming only, and other feeding structures such as the labrum are also highly reduced. In the antennulae, the morphological changes in form and setation were followed from nauplius to cypris and shown to largely resemble events in ontogeny of the thoracican barnacle Semibalanus balanoides. On this basis we establish a homology scheme between antennulary setae in these two species. In both B. tenellus and S. balanoides, a naupliar seta, apically on the fourth antennulary segment develops into a conspicuous aesthetasc while one (B. tenellus) or two (S. balanoides) subapical and postaxially sited setae on the same segment develops into into four short setae in the cypris. Our study reveals that the terminology used in describing cirripede nauplius and cypris larvae is often misleading or even erroneous and improvements are suggested. Notably replacing cypris carapace with the ontogenetically and phylogenetically more informative term head shield.     

Foreseeing fates: a commentary on Manton (1928) ‘On the embryology of a mysid crustacean,Hemimysis lamornae’

Sidnie Manton became best known for her work on arthropod locomotion, and for proposing radical views on the evolution of arthropods that were accepted for a generation. However, her early training was as an embryologist, and the work that she carried out at the beginning of her career still stands as one of the major twentieth century contributions to the study of crustacean embryology. Here, I review her first major paper, largely completed while she was a graduate student, describing embryonic development in Hemimysis lamornae, a small shrimp-like animal found in the seas around the UK. The clarity of her writing and the quality of her figures set a standard that laid the basis for subsequent work, and although not all of her conclusions have stood the test of time, they remain a standard reference for work today. This commentary was written to celebrate the 350th anniversary of the journal Philosophical Transactions of the Royal Society.     

The chelifores of sea spiders (Arthropoda,Pycnogonida) are the appendages of the deutocerebral segment

SUMMARY Within the last decade, gene expression patterns and neuro‐anatomical data have led to a new consensus concerning the long‐debated association between anterior limbs and neuromeres in the arthropod head. According to this new view, the first appendage in all extant euarthropods is innervated by the second neuromere, the deutocerebrum, whereas the anterior‐most head region bearing the protocerebrum lacks an appendage. This stands in contrast to the clearly protocerebrally targeted “antennae” of Onychophora and to some evidence for protocerebral limbs in fossil euarthropod representatives. Yet, the latter “frontal appendages” or “primary antennae” have most likely been reduced or lost in the lineage, leading to extant taxa. Surprisingly, a recent neuro‐anatomical study on a pycnogonid challenged this evolutionary scenario, reporting a protocerebral innervation of the first appendages, the chelifores. However, this interpretation was soon after questioned by Hox gene expression data. To re‐evaluate the unresolved controversy, we analyzed neuro‐anatomy and neurogenesis in four pycnogonid species using immunohistochemical techniques. We clearly show the postprotocerebral innervation of the chelifores, which is resolved as the plesiomorphic condition in pycnogonids when evaluated against a recently published comprehensive phylogeny. By providing direct morphological support for the deutocerebral status of the cheliforal ganglia, we reconcile morphological and gene expression data and argue for a corresponding position between the anterior‐most appendages in all extant euarthropods. Consequently, other structures have to be scrutinized to illuminate the fate of a presumptive protocerebral appendage in recent euarthropods. The labrum and the “frontal filaments” of some crustaceans are possible candidates for this approach.